There are limited options for reconstruction of bone defects resulting from congenital anomaalies, trauma, infection, and oncologic resection. Nearly one million bone graft procedures are performed annually, with the clinical 'gold standard' being the use of decellularized allografts. Of these allograft implantation procedures, nearly 60% fail within 10 years of implantation due to poor graft-host integration and microcrack propagation. Unlike allografts, autografts fully heal and integrate, mediated by the periosteum, a thin layer of tissue and periosteal cells (PCs) surrounding bone, where healing is coordinated by a variety of contextual cues including matrix and paracrine factors. PCs, which persist during autografts healing for only ~21 days, are phenotypically similar to bone marrow-derived mesenchymal stem cells (MSCs). Therapeutically, however, MSCs are favored compared to PCs as they are isolated from bone marrow, reducing bone tissue morbidity resulting from PC isolation. A critical knowledge gap exists in identifying the critical cues (paracrine factors, matrix interactions, etc. that orchestrate autograft healing and are absent in allografts, preventing the translation of therapies to effectively revitalize allografts. Our objective is to develop periosteum mimetics composed of synthetic hydrogels (poly(ethylene glycol), PEG) for MSC transplantation to (1) promote cell-mediated allograft healing/integration, to (2) isolate the critical factors of the periosteum in healing, and to (3) develop cell-free therapies that result in complete allograft healing and integration. Hydrogels will be used to surround allografts, taking advantage of structural integrity of allografts and improving what is insufficient in healing and integration by recreating the periosteum. We hypothesize that hydrogel nanoarchitectures can be tuned through alterations in degradation and biochemical functionalities to promote MSC-mediated allograft healing and integration. We further hypothesize that MSCs promote healing through simple release of paracrine factors, thus, cell-free revitalization approaches can be developed. The rationale for this work is to identify translatable therapies, based on critical healing factor, to improve healing and integration of the 300,000 massive allograft procedures performed annually in the US. Three specific aims are outlined: Aim 1: Develop periosteum- mimetic PEG hydrogels to support MSC-mediated allograft healing in vivo. Aim 2: Identify critical paracrine factors produced by hydrogel-transplanted MSCs that modulate allograft healing. Aim 3: Develop paracrine factor-releasing hydrogels to enhance allograft revitalization in the absence of cell transplantation. Successful completion of these Aims will significantly advance our understanding of how MSCs coordinate allograft healing and integration and of how to design synthetic polymer scaffolds to promote natural bone regeneration processes. This material platform should be readily tailored for applications towards regenerating tissues beyond bone, as well as providing specific advantages for future directions in the design of cell delivery vehicles. !